Solid-Phase Synthesis and Combinatorial Technologies

a: DP3/EP3 (0.5/0.5 eqs.), DCM, reflux; b: HCOOH, rt; c: AP3/BP3/DP3 (0.375/0.25/0.375 eqs.), rt.

Figure 11.18 Structures of isocyanates AP3-EP3 and synthesis of the solution-phase hybrid dendrimers 11.15–11.17.

tionalization, which may lead to great variability in physicochemical properties exploitable in disciplines such as catalysis and phase-transfer agents (93).

11.3.3 Polymer Libraries as Sources of Reagents

The use of polymeric reagents or catalysts is popular in organic synthesis. Usually, the reactive entity is attached to a solid support, and while its chemical properties are similar to its solution counterpart, the heterogeneicity of the solid supported version

604 MATERIALS AND POLYMERIC COMBINATORIAL LIBRARIES

allows easier work-up/purification procedures, or stabilizes the supported reactant, or simplifies the recovery of precious catalysts. A general overview of supported reagents and catalysts, with particular attention to their application in solution-phase combinatorial library synthesis, was reported in Section 8.4.

Menger et al. (97) presented an alternative approach, where an 8198-member discrete polymer library L14 was prepared and tested as a source of reducing agents for the ketone–alcohol transformation in aqueous media. The library features are reported in Fig. 11.19. Its synthesis was carried out using an automated liquid dispenser to deliver and withdraw the solutions of reagents. Two polymeric amines 11.18 and 11.19 were reacted with mixtures of three or four acids selected from the monomer set M1 (15 representatives, Fig. 11.19). The coupling was performed with a substoichiometric mixture of acids (typically 20–60% of the polymer amine sites), leaving a substantial amount of free amino groups. The final structure of L14 was obtained by functionalization of 5–10% of the loading sites with dihydropyridine moieties (DHP; 25–100 DHP units per polymer molecule) during the amide coupling. Known biological systems (98, 99), based on the oxidation of DHP moieties and the concomitant reduction of ketones to alcohols, suggested DHP as a potential polymer-supported ketone reducing agent in aqueous media.The presence of metal ions (Cu2+, Mg2+, or Zn2+) was also required for the reducing activity. Two randomly selected library individuals with different polyamine scaffolds could be represented as 11.20 and 11.21 (Fig. 11.19), where the percentages refer to the quantity of each acid versus the number of amine loading sites. The exact determination of functionalized versus nonfunctionalized sites and the relative location of each acid molecule on the polymer backbone was obviously impossible. Each library individual, thus, was more precisely a mixture of structural isomers containing the same relative amounts of each monomer.

The selected test reduction of benzoyl formic acid 11.22 to racemic mandelic acid 11.23 was performed under carefully optimized reaction conditions (Fig. 11.20). The reaction was monitored by following the reduction of intensity of the DHP chromophore at 340 nm as it was oxidized, and the yields for each library individual were expressed as the percentage of oxidized DHP sites per polymer molecule. Yields varied from 0 to 50%, with average reaction times of 4 h. The accuracy of the correspondence between this value and the amount of recovered reaction product 11.23 was checked, performing the large-scale synthesis of a specific active library individual and purifying the obtained 11.23 by chromatography. The screening produced around 25 composites (0.3%) with a conversion yield of >40%. The structures and the reducing activity of several library individuals (11.24–11.31) are reported in Table 11.3.

The 10 best polymers were reprepared in larger amounts to confirm their properties, and each preparation was repeated twice to check if the random disposition of the acid-derived functional groups on the polyamine backbone was influencing the reducing efficiency. All the polymers were confirmed as active, and the two batches showed comparable activities, confirming the reproducibility of this synthetic method. Several crude indications in terms of SAR were identified, and a structural specificity of reducing efficiency was clearly present, albeit difficult to rationalize. For example, compare the activity of similar composites 11.27 (40% yield) and 11.29 (1.3% yield). Many modifications of the polymeric scaffold (different average MWs, increase of

10% Thi-2;

10% Zn2+.

Figure 11.19 Structure of the monomer set M1 used for the synthesis of the polyamine amide catalyst library L14 and of the library individuals 11.20 and 11.21.

DHP loading) or of the experimental reaction protocols (presence of cosolvents, absence of metal ions) completely canceled the reducing efficiency.

The cheap, commercially available precursors as well as the simplicity of the synthesis, characterization, and use of such a polymer as a reagent make this approach

5% Cap.

11.35

Figure 11.21 Structure of the monomer set M1 used for the synthesis of the polyacid amide artificial enzyme library L15 and the library individual 11.35.

absorbance of a pure, independently prepared solution of the product 11.34. Several library individuals (<1% of L15) showed an efficient catalysis of the dehydration reaction, as, for example, 11.35 (Fig. 11.21). An average 1000-fold enhancement of the uncatalyzed dehydration reaction rate was estimated. The reliability and reproduci-

608 MATERIALS AND POLYMERIC COMBINATORIAL LIBRARIES

bility of the results was confirmed, as in the previous example, by larger repreparations of multiple batches of active library individuals.

An interesting finding was a common lag period of around 24 h for each catalyst, which was followed for the active library individuals by a fast and efficient catalytic cycle. This catalytic activity was eradicated by low (0 °C) or high (40 °C) reaction temperatures, by sonication of the reaction mixture, or by cross-linking and stiffening of the polymeric matrix. A hypothesis in keeping with all the experimental findings is the slow substrate-induced modification of the polymer into a catalytically active conformation, which could have been disrupted by sonication or higher temperature/energy, or prevented by the rigid structure of the cross-linked matrix, or by the lower temperature/energy.

Menger et al. (33) also reported a similar polymeric library to identify catalysts for the hydrolysis of a phosphate ester (102), where a 30,000-fold increase versus the uncatalyzed reaction rate was obtained. Miller and Ford (103) reported the synthesis of a 32-member discrete polymer library based on anion-exchange latexes and its screening for the alkaline hydrolysis of p-nitrophenylalkane carboxylates. The reported efforts may represent just the tip of an iceberg in terms of opportunities granted by polymeric catalysts, and an expansion of knowledge derived from further efforts is to be expected in the near future.

11.3.5 Polymer Libraries as Sources of Biodegradable Medical Implants

The use of combinatorial technologies in pharmaceutical research has been vastly limited to the early phases of the process of drug discovery (see Section 9.1), while later phases in the development of a drug and medical applications of library individuals have always been considered exempt from a combinatorial way of thinking. In fact, their synthetic throughput is usually low, because the efforts are extremely focused and parallel/combinatorial approaches are not considered necessary.

Brocchini et al. (104, 105) reported an intriguing application of parallel synthesis for the fast identification and characterization of polymeric materials suitable for biomedical applications. An 112-member focused polymer library L16 was prepared and screened as a source of suitable materials for medical implants. Its features and the structure of the monomer sets M1 (14 diphenols) (106) and M2 (8 diacids) are reported in Fig. 11.22. The library was designed using degradable, biocompatible monomers to help stimulate the regrowth of functional tissue in the human body. An extensive amount of available information regarding similar structures was used to help select the building blocks and the properties to be monitored for each library individual (vide infra). The synthetic protocol was adapted from reported procedures (106, 107) and was carefully set up to accommodate a higher synthetic throughput together with a high control of the polymeric reaction products. The monomer representatives were selected among candidates that exhibited similar reactivity during monomer rehearsal, and the parallel experimental procedures (stirring, solvent delivery, etc.) were regulated to obtain an average quantity of 200 mg of each library component. Careful weighing of monomers was required to ensure their stoichiometric equivalence and thus the high quality of final library individuals.

X= CH2 or CH2CH2 (see M1)

Y= see M2

R = see M1

X = CH2; R = Me, Et, i-Pr, n-Bu, i-Bu, s-Bu, n-Hex, n-Oct, n-Dodecyl, benzyl, EtOEtOEt X = CH2CH2; R = Et, n-Hex, n-Oct

Y = (CH2)3, (CH2)2, (CH2)4, (CH2)6, (CH2)8, CH2CH(CH3)CH2CH2, CH2OCH2, CH2OCH2CH2OCH2

Figure 11.22 Structure of the monomer sets M1 and M2 used to prepare the biocompatible polymer amide ester library L16.

The materials were characterized using classical polymer science techniques, with less than 40 mg of each sample being required for the measurement of their most relevant physicochemical properties. They all proved to be stable amorphous solids (decomposition above 300 °C), and all were suitable for larger scale production. Their high MW (50,000 < MW < 150,000) and their polymer polydispersity index (from 1.4 to 2) were also appropriate for use as medical implants. Despite their structural similarity, the different backbones and pendent chains produced both stiff and flexible individuals with low (down to 2 °C) or high (up to 91 °C) glass transition temperatures and with varying air–water contact angles (from 64° to 101°). The 112 library individuals were thus similar enough to biomedically relevant models to represent suitable potential candidates for medical applications, but they were also different enough to deserve testing and screening to build an SAR and to eventually select the best candidates for different applications.

A sublibrary L17, made of 42 diverse library individuals (Fig. 11.23), was screened in an in vivo model for the proliferation of fibroblasts, where a high value represented a candidate for the tissue reconstruction process around the medical implant. The screening protocol was performed in parallel, and a correlation was established between the fibroblast proliferation (biological property) and the air–water contact angle (physicochemical property). A crude SAR was assessed for further, more

610 MATERIALS AND POLYMERIC COMBINATORIAL LIBRARIES

L17

42-member discrete sublibrary

of L16

R

X = CH2; R = Me, Et, n-Bu, n-Hex, n-Oct, n-Dodecyl

Y = (CH2)3, (CH2)2, (CH2)4, (CH2)6, (CH2)8, CH2OCH2, CH2OCH2CH2OCH2

Figure 11.23 Structure of the monomer sets M1 and M2 used to prepare the L16-derived, biocompatible polymer amide ester sublibrary L17.

focused efforts, and several composites were considered appealing candidates for realizing novel medical implant devices. Several other significant findings were obtained from the analysis of L16 and L17, and the potential applicability of other polymers in other fields of polymer science was mentioned by the authors.

The application of combinatorial technologies, or better combinatorial/parallel protocols, to more classical, downstream projects, including in vivo testing such as the example reported above, has the potential to significantly decrease the time needed for information gathering and to better rationalize largely untouched areas. The largely diffused belief that the usefulness of combinatorial technologies is limited to the early research phases should be disproven more and more by similar examples.

11.3.6 Molecularly Imprinted Polymer Libraries

Molecularly imprinted polymers (MIPs) (108, 109) have recently emerged as a class of artificial receptors with desirable properties. They are produced by copolymerization of functional monomers and cross-linking agents in the presence of a template molecule, which drives the polymerization so as to maximize the noncovalent interactions between the polymer backbone and the template (Fig. 11.24; compare the template-driven path b with the random process of path a). Simple removal of the soluble template from the cavities in the polymer structure leaves a template-specific imprint. The resulting MIPs may display high affinities for the template and, when compared to biological antibodies or receptors, possess a higher stability and can be handled safely. They have found applications in many disciplines, and it is easy to predict an exponential growth of interest in MIPs from the scientific community. There

POLYMERIZATION

4 FUNCTIONAL MONOMERS

path a

PLUS MANY OTHER

POSSIBILITIES

path b

TEMPLATE-ASSISTED

POLYMERIZATION

MOLECULARLY

IMPRINTED

POLYMER

TEMPLATE

Figure 11.24 Molecularly imprinted polymers (MIP): random (top) and template-assisted (bottom) polymerization process.

are, however, a number of drawbacks that are limiting MIP-related research, such as the typically long process (up to several days) for their preparation and characterization and the absence of assessed SARs/established information databases able to drive the rational design of MIPs for a specific purpose.